Acid-labile lipophilic prodrugs of cancer chemotherapeutic agents

ABSTRACT

The present application discloses an acid labile lipophilic molecular conjugate of cancer chemotherapeutic agents and methods for reducing or substantially eliminating the side effects of chemotherapy associated with the administration of a cancer chemotherapeutic agent to a patient in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Non-Provisional applicationSer. No. 13/856,216, filed Apr. 3, 2013, which claims the benefit ofU.S. Non-Provisional application Ser. No. 13/489,247, filed Jun. 5,2012, which claims the benefit of U.S. Provisional Application No.61/493,827 filed Jun. 6, 2011 and U.S. Provisional Application No.61/496,367 filed Jun. 13, 2011, the full contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to chemical compounds andmethods for use in treating patients. More particularly, the presentinvention is directed to molecular conjugates for use in cancertreatment. Specifically, the present invention relates to acid-labile,lipophilic conjugates, methods and intermediates useful in the formationthereof, and methods for treating a patient therewith.

BACKGROUND OF THE INVENTION

A number of anti-cancer drug are currently in the market for thetreatment of various cancers. For example, paclitaxel and docetaxel aretwo promising anti-cancer drugs used to treat breast and ovariancancers, and which hold promise for the treatment of various othercancers such as skin, lung, head and neck carcinomas. Other promisingchemotherapeutic agents are being developed or tested for treatment ofthese and other cancers. Compounds such as paclitaxel, docetaxel andother taxanes, camptothecins, epothilones and quassinoids, as well asother compounds exhibiting efficacy in cancer treatment, are ofconsiderable interest. Of special interest are natural product drugs andtheir synthetic analogs with demonstrated anticancer activity in vitroand in vivo.

However, many identified anti-cancer compounds present a number ofdifficulties with their use in chemotherapeutic regimens. A majorproblem with the use of such chemotherapeutic agents in cancer treatmentis the difficulty targeting cancer tissues, without adversely affectingnormal, healthy tissues. For example, paclitaxel exerts its antitumoractivity by interrupting mitosis and the cell division process, whichoccurs more frequently in cancer cells, than in normal cells.Nonetheless, a patient undergoing chemotherapy treatment may experiencevarious adverse effects associated with the interruption of mitosis innormal, healthy cells.

Targeted cancer therapies that can selectively kill cancer cells withoutharming other cells in the body would represent a major improvement inthe clinical treatment of cancer. Reports of targeting drugs usingantibodies have appeared in the literature since 1958. Targeting drugsby conjugation to antibodies for selective delivery to cancer cells hashad limited success due to the large size of antibodies (MW=125-150kilodaltons) and thus their relative inability to penetrate solidtumors.

An alternative strategy comprises the use of smaller targeting ligandsand peptides, which recognize specific receptors unique to oroverexpressed on tumor cells, as the targeting vector. Such constructshave molecular weights of 2-6 kilodaltons, which allow ready penetrationthroughout solid tumors.

Accordingly, it would be highly desirable to develop novel compounds andmethods for use in directly targeting cancer cells with chemotherapeuticagents in cancer treatment regimens. This, in turn, could lead toreduction or elimination of toxic side effects, more efficient deliveryof the drug to the targeted site, and reduction in dosage of theadministered drug and a resulting decrease in toxicity to healthy cellsand in the cost of the chemotherapeutic regimen.

One particular approach of interest is the use of anticancer drugmoieties that have been conjugated to tumor molecules. For example, U.S.Pat. No. 6,191,290 to Safavy discusses the formation and use of a taxanemoiety conjugated to a receptor ligand peptide capable of binding totumor cell surface receptors. Safavy in particular indicates that suchreceptor ligand peptides might be a bombesin/gastrin-releasing peptide(BBN/GRP) receptor-recognizing peptide (BBN [7-13]), a somatostatinreceptor-recognizing peptide, an epidermal growth factorreceptor-recognizing peptide, a monoclonal antibody or areceptor-recognizing carbohydrate.

One important aspect of synthesizing these drug molecular conjugates isconnecting these two units with a linker or linkers that provideconjugates with desired characteristics and biological activity, inparticular, a conjugate that is stable in systemic circulation butreleases cytotoxic agent once internalized into cancer cells orconcentrated in the locally acidic tumor environment. Such an agentwould be expected to exhibit lower toxicity to normal tissues. Theresulting conjugate should also be sufficiently stable until it reachesthe target tissue, and thus maximizing the targeting effect with reducedtoxicity to normal, healthy tissue.

The blood-brain barrier (BBB) is a specialized physical and enzymaticbarrier that segregates the brain from systemic circulation. Thephysical portion of the BBB is composed of endothelial cells arranged ina complex system of tight junctions which inhibit any significantparacellular transport. The BBB functions as a diffusion restraintselectively discriminating against substance transcytosis based on lipidsolubility, molecular size and charge thus posing a problem for drugdelivery to the brain. Drug delivery across the BBB is furtherproblematic due to the presence of a high concentration of drug effluxtransporters (e.g., P-glycoprotein, multi-drug resistant protein, breastcancer resistant protein). These transporters actively remove drugmolecules from the endothelial cytoplasm before they even cross into thebrain.

The methods that are currently employed for drug delivery in treatmentof brain malignancies are generally nonspecific and inefficient. Anadditional problem to consider when treating brain diseases is thediffusion of the drug in its vehicle across the tumor or affectedtissue. Mostly the size, as well as other physiologic characteristics ofthe vehicles that are currently in use for such delivery of drugs to thebrain, hamper efficient diffusion of the drug through the diseasedtissue. The lack of efficient drug diffusion affects the efficacy of thetreatment.

Peptides have been extensively studied as carrier molecules for drugdelivery to the brain in hope they could be employed as drug deliveryvehicles. Peptides are, however, problematic due to their limitedbioavailability. Even though methods to increase the bioavailability ofsuch molecules have been intensively explored, they resulted in modestsuccess at best.

Increased cell proliferation and growth is a trademark of cancer. Theincrease in cellular proliferation is associated with high turnover ofcell cholesterol. Cells requiring cholesterol for membrane synthesis andgrowth may acquire cholesterol by receptor mediated endocytosis ofplasma low density lipoproteins (LDL), the major transporter ofcholesterol in the blood, or by de novo synthesis.

LDL is taken up into cells by a receptor known as the LDL receptor(LDLR); the LDL along with the receptor is endocytosed and transportedinto the cells in endosomes. The endosomes become acidified and thisreleases the LDL receptor from the LDL; the LDL receptor recycles to thesurface where it can participate in additional uptake of LDL particles.There is a body of evidence that suggests that tumors in a variety oftissues have a high requirement for LDL to the extent that plasma LDLsare depleted. The increased import of LDL into cancerous cells isthought to be due to elevated LDL receptors (LDLR) in these tumors. Sometumors known to express high numbers of LDLRs include some forms ofleukemia, lung tumors, colorectal tumors and ovarian cancer.

Comparative studies of normal and malignant brain tissues have shown ahigh propensity of LDLRs to be associated with malignant and/or rapidlygrowing brain cells and tissues. Some studies suggest that rapidlygrowing brain cells such as those seen in early development and inaggressively growing brain tumors exhibit increased expression of LDLRsdue to their increased requirement for cholesterol.

Among the problematic and inefficiently treated brain cancers isglioblastoma multiforme (GBM). This devastating brain tumor is 100%fatal. Moreover, over 85% of total primary brain cancer-related deathsare due to GBM. Current therapies rely on a multimodal approachincluding neurosurgery, radiation therapy and chemotherapy. Even thebest efforts using these approaches have resulted in only a modestincrease in survival time for patients afflicted with this tumor.

GBM being gliomas of the highest malignancy is characterized byuncontrolled, aggressive cell proliferation and general resistance toconventional therapies. GBM cells in culture have high numbers of lowdensity lipoprotein receptors (LDLR). Since this receptor is nearlyabsent in neuronal cells and normal glial cells, it represents an idealtarget for the delivery of therapeutic agents such as cytotoxins orradiopharmaceuticals. Efforts to improve existing therapies or todevelop new ones have not been successful and the outcome of treatmentfor malignant gliomas is only modest, at best, with a median survivaltime of approximately 10 months.

Unlike normal brain cells that have few LDL receptors, GBM cells inculture have high numbers of LDL receptors on their surface. Othercancers are likely to also have high expression of LDLR due to thehighly proliferative nature of the cancerous tissue and need forcholesterol turnover. This suggests that the LDL receptor is a potentialunique molecular target in GBM and other malignancies for the deliveryof anti-tumor drugs via LDL particles.

Maranhão and coworkers have demonstrated that a cholesterol-richmicroemulsion or nanoparticle preparation termed LDE concentrates incancer tissues after injection into the bloodstream. D. G. Rodrigues, D.A. Maria, D. C. Fernandes, C. J. Valduga, R. D. Couto, O. C. Ibanez andR C. Maranhão. Improvement of paclitaxel therapeutic index byderivatization and association to a cholesterol-rich microemulsion: invitro and in vivo studies. Cancer Chemotherapy and Pharmacology 55:565-576 (2005). The cytotoxicity, pharmacokinetics, toxicity to animalsand therapeutic action of a paclitaxel lipophilic derivative associatedto LDE were compared with those of commercial paclitaxel. Results showedthat LDE-paclitaxel oleate was stable. The cytostatic activity of thedrug in the complex was diminished compared with the commercialpaclitaxel due to the cytotoxicity of the vehicle Cremophor EL used inthe commercial formulation. Maranhão and coworkers showed LDE-paclitaxeloleate is a stable complex and compared with paclitaxel, toxicity isconsiderably reduced and activity is enhanced which may lead to improvedtherapeutic index in clinical use.

Capturing the great potential of selective and specific delivery ofchemotherapeutic compounds to cancer tissues via their over expressionof LDL receptors and consequent high uptake of LDL particles from thesystemic circulation, requires that the cancer chemotherapeutic agenthave high lipophilicity so as to remain entrapped in the lipid core ofthe LDL particle and not diffuse into the plasma to lead to toxic sideeffects from exposure of normal tissues to the agent. Further, once theLDL particle with its chemotherapeutic payload has entered the cancercell via LDL receptor mediated uptake into the acidic environment of theendosome, the LDL receptor is disassociated from the LDL particle and isrecycled to the cell surface and the LDL particle releases its lipidcontents and its lipophilic chemotherapeutic agent to the enzymes andacidic environment of the endosome. Few cancer chemotherapeutic agentsare intrinsically sufficiently lipophilic to be retained adequatelywithin the lipid core of the LDL particle. This creates a need forsuitable lipophilic derivatives of the cancer chemotherapeutic agentwhich have high stability in normal systemic circulation and retentionin the lipid core of the LDL particles but readily release the activechemotherapeutic agent in the acidic environment of the endosome. Thecompounds of the present invention address this need.

Definitions

As used herein, the term “alkyl”, alone or in combination, refers to anoptionally substituted straight-chain or branched-chain alkyl radicalhaving from 1 to 22 carbon atoms (e.g. C₁-C₂₂ alkyl or C₁₋₂₂ alkyl).Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, tert-amyl, pentyl, hexyl,heptyl, octyl and the like. In certain embodiments, the alkyl group,such as a C₁-C₂₂ alkyl or C₅-C₂₂ alkyl, may also include one or moredouble bonds in the alkyl group, and may also referred to as in a C₁-C₂₂alkenyl or C₅-C₂₂ alkenyl group.

The term “alkenyl”, alone or in combination, refers to an optionallysubstituted straight-chain or branched-chain hydrocarbon radical havingone or more carbon-carbon double-bonds and having from 2 to about 22carbon atoms. Examples of alkenyl radicals include ethenyl, propenyl,1,4-butadienyl and the like.

The term “alkoxy” refers to an alkyl ether radical wherein the termalkyl is defined as above. Examples of alkoxy radicals include methoxy,ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy,tert-butoxy and the like.

The term “diastereoisomer” refers to any group of four or more isomersoccurring in compounds containing two or more asymmetric carbon atoms.Compounds that are stereoisomers of one another, but are not enantiomersare called diastereoisomers.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. Protectinggroup chemistry has been reviewed (Greene, T. W.; Wuts, P. G. M.Protective Groups in Organic Synthesis, 4th ed.; Wiley: New York, 2007).Exemplary silyl groups for protection of hydroxyl groups include TBDMS(tert-butyldimethylsilyl), NDMS (2-norbornyldimethylsilyl), TMS(trimethylsilyl) and TES (triethylsilyl). Exemplary NH-protecting groupsinclude benzyloxycarbonyl, t-butoxycarbonyl and triphenylmethyl.

The terms “taxanes,” “taxane derivatives,” and “taxane analogs” etc . .. are used interchangeably to mean compounds relating to a class ofantitumor agents derived directly or semi-synthetically from Taxusbrevifolia, the Pacific yew. Examples of such taxanes include paclitaxeland docetaxel and their natural as well as their synthetic orsemi-synthetic derivatives.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablesalts” as used herein, means the excipient or salts of the compoundsdisclosed herein, that are pharmaceutically acceptable and provides thedesired pharmacological activity. These excipients and salts includeacid addition salts formed with inorganic acids such as hydrochloricacid, hydrobromic acid, phosphoric acid, and the like. The salt may alsobe formed with organic acids such as acetic acid, propionic acid,hexanoic acid, glycolic acid, lactic acid, succinic acid, malic acid,citric acid, benzoic acid and the like.

“Therapeutically effective amount” means a drug amount that elicits anyof the biological effects listed in the specification.

SUMMARY OF THE INVENTION

In one embodiment, there is provided new and useful compositions ofmolecular conjugates of hydroxyl-bearing cancer chemotherapeutic agents(HBCCA). In another embodiment, there is provided compositions of acidlabile, lipophilic molecular conjugates of cancer chemotherapeuticagents for use in treating cancer. In another embodiment, there isprovided intermediate compounds for use in forming molecular conjugates,such as acid labile, lipophilic pro-drug conjugates, for use in treatingcancer. In another embodiment, there is provided efficient methods forthe preparation of acid labile, lipophilic drug conjugates. In anotherembodiment, there is provided methods for administering chemotherapeuticagents to patients that reduce or substantially eliminate side effectsconventionally experienced by cancer patients. In another embodiment,there is provided methods for concentrating chemotherapeutic agents incancer cells of a patient.

In one embodiment, there is provided an acid labile lipophilic molecularconjugate (ALLMC) of the formula 1, 1.1 or formula 2:

wherein: R is a hydroxyl bearing cancer chemotherapeutic agent; forformula 1 or 1.1 R¹ is hydrogen, C₁-C₄ alkyl or C₅-C₂₂ alkyl; R² isC₅-C₂₂ alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen orC₁-C₆ alkyl; Z is O or S; Q is O or S; and T is O or S; for formula 2:R² is a C₁-C₂₂ alkyl; T is O or S; and X is hydrogen or a leaving groupselected from the group consisting of mesylates, sulfonates and halogen(Cl, Br and I); and their isolated enantiomers, diastereoisomers ormixtures thereof, or a pharmaceutically acceptable salt thereof. Thecompound 1.1 includes the pure syn isomer, the pure anti isomer andmixtures of syn- and anti-isomers, and their diastereomers.

In another embodiment, there is provided the above acid labilelipophilic molecular conjugate of the formula 1 or 1.1 wherein: R is ahydroxyl bearing cancer chemotherapeutic agent; R¹ is hydrogen, C₁-C₄alkyl or C₅-C₂₂ alkyl; R² is C₅-C₂₂ alkyl; Y is O or S; Z is O; Q is O;and T is O. In one aspect of the acid labile lipophilic molecularconjugate of the formula 2 wherein: R² is C₅-C₂₂ alkyl; T is O; and X ishydrogen or selected from the group consisting of Cl, Br and I. Inanother variation, R² is C₉-C₂₂. In another aspect of the above acidlabile lipophilic molecular conjugate comprising the formula 1a, 1b orformula 2a:

wherein: R is a hydroxyl bearing cancer chemotherapeutic agent (HBCCA);for formula 1a or 1b R¹ is hydrogen, C₁-C₄ alkyl or C₅-C₂₂ alkyl; and R²is C₅-C₂₂ alkyl; andfor formula 2a: R² is C₁-C₂₂ alkyl; and X is hydrogen or is selectedfrom the group consisting of Cl, Br and I. In one variation of thecompound that is the carbonate (i.e., —OC(O)O—) of the formula 1a or 1bthe compound is the corresponding sulfonate (i.e., —OS(O)O—) of theformula 1a wherein the carbonate group is replaced by a sulfonate group.The compound 1b includes the pure syn isomer, the pure anti isomer andmixtures of syn and anti isomers, and their diastereomers.

In another variation of the compound of the formula 1, 2, 1a and 2a, R¹is hydrogen or C₁-C₄ alkyl or C₅-C₂₂ alkyl, and R² is the carbon residueof an unsaturated fatty acid, such as the carbon residue selected fromthe group consisting of the C₁₉ residue of eicosenoic acid (includingthe cis isomer, trans isomer and mixtures of isomers), C₁₇ residue ofoleic acid and the C₁₇ residue of elaidic acid. As used herein, the“carbon residue” (e.g., C₁₇ residue, C₁₉ residue etc. . . . ) of thefatty acid means the carbon chain of the fatty acids excluding thecarboxyl carbon.

In another aspect of the above acid labile lipophilic molecularconjugate, the hydroxyl bearing cancer chemotherapeutic agent isselected from the group consisting of taxanes, abeo-taxanes,camptothecins, epothilones, cucurbitacins, quassinoids, anthracyclines,and their analogs and derivatives. In another aspect of the above acidlabile lipophilic molecular conjugate, the hydroxyl bearing cancerchemotherapeutic agent is selected from the group consisting ofaclarubicin, camptothecin, masoprocol, paclitaxel, pentostatin,amrubicin, cladribine, cytarabine, docetaxel, gemcitabine, elliptiniumacetate, epirubicin, etoposide, formestane, fulvestrant, idarubicin,pirarubicin, topotecan, valrubicin and vinblastine. In another aspect ofthe above acid labile lipophilic molecular conjugate, the conjugate isselected from the compounds in FIGS. 18, 19 and 20. In one variation,only one of the groups -ALL¹, -ALL², -ALL³ . . . to -ALL^(n) is an -ALLgroup and the others are hydrogens. In another variation, two of thegroups -ALL¹, -ALL², -ALL³ . . . to -ALL^(n) are -ALL groups.

In another embodiment, there is provided a pharmaceutical compositioncomprising: a) a therapeutically effective amount of a compound of theabove, in the form of a single diastereoisomer; and b) apharmaceutically acceptable excipient. In another aspect, thepharmaceutical composition is adapted for oral administration; or as aliquid formulation adapted for parenteral administration. In anotheraspect, the composition is adapted for administration by a routeselected from the group consisting of orally, parenterally,intraperitoneally, intravenously, intraarterially, transdermally,intramuscularly, rectally, intranasally, liposomally, subcutaneously andintrathecally. In another embodiment, there is provided a method for thetreatment of cancer in a patient comprising administering to the patienta therapeutically effective amount of a compound or composition of anyof the above compound or composition, to a patient in need of suchtreatment. In one aspect of the method, the cancer is selected from thegroup consisting of leukemia, neuroblastoma, glioblastoma, cervical,colorectal, pancreatic, renal and melanoma. In another aspect of themethod, the cancer is selected from the group consisting of lung,breast, prostate, ovarian and head and neck. In another aspect of themethod, the method provides at least a 10%, 20%, 30%, 40%, or at least a50% diminished degree of resistance expressed by the cancer cells whencompared with the non-conjugated hydroxyl bearing cancerchemotherapeutic agent.

In another embodiment, there is provided a method for reducing orsubstantially eliminating the side effects of chemotherapy associatedwith the administration of a cancer chemotherapeutic agent to a patient,the method comprising administering to the patient a therapeuticallyeffective amount of an acid labile lipophilic molecular conjugate of theformula 1, 1.1 or formula 2:

wherein: R is a hydroxyl bearing cancer chemotherapeutic agent; forformula 1 or 1.1: R¹ is hydrogen, C₁-C₄ alkyl or C₅-C₂₂ alkyl; R² isC₅-C₂₂ alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen orC₁-C₆ alkyl; Z is O or S; Q is O or S; and T is O or S; for formula 2:R² is C₁-C₂₂ alkyl; T is O or S; and X is hydrogen or a leaving groupselected from the group consisting of mesylates, sulfonates and halogen(Cl, Br and I); and their isolated enantiomers, diastereoisomers ormixtures thereof. The compound 1.1 includes the pure syn isomer, thepure anti isomer and mixtures of syn and anti isomers, and theirdiastereomers. In one variation of the above, R² is C₉-C₂₂ alkyl. In oneaspect, the method provides a higher concentration of the cancerchemotherapeutic agent in a cancer cell of the patient. In anotheraspect, the method delivers a higher concentration of the cancerchemotherapeutic agent in the cancer cell, when compared to theadministration of a non-conjugated cancer chemotherapeutic agent to thepatient, by at least 5%, 10%, 20%, 30%, 40% or at least 50%.

In another embodiment, there is provided a compound of the formula 3a or3b:

wherein: R¹ is hydrogen, C₁-C₄ alkyl or C₅-C₂₂ alkyl; R² is C₅-C₂₂alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen or C₁-C₆alkyl; Z is selected from O or S; Q is O or S; and T is O or S. In oneaspect of the compound, R¹ is hydrogen or C₁-C₄ alkyl; R² is C₅-C₂₂alkyl; Y is O or S; Z is O; Q is O; and T is O. The activated compoundof the formula 3a or 3b may be used to prepare the acid labilelipophilic conjugate when the activated compound is condensed with ahydroxyl bearing cancer chemotherapeutic agent (HBCCA). As definedherein, the HBCCA is represented generically with the residue or group“R” in the formulae 1, 1a, 1b, 1.1 and 2a, for example, and where theHBCCA is not coupled to form the acid labile, lipophilic molecularconjugates, then the HBCCA may also be generically represented as havingthe formula “R—OH” since the HBCCA may be functionalized by one or morehydroxyl (—OH) groups. Similarly, the acid labile lipophilic group(i.e., the “-ALL” group of the activated compound) that may be condensedwith a HBCCA to form the acid labile, lipophilic molecular conjugategenerically represented as “R—O-ALL.” Accordingly, where more than one-ALL group is condensed or conjugated with a HBCCA group, then each -ALLgroup may be independently designated as -ALL¹, -ALL², -ALL³ . . . to-ALL^(n) where n is the number of available hydroxyl groups on thecancer chemotherapeutic agent that may be conjugated or couple with an-ALL group. As exemplified for the compound of formulae 1 and 2, forexample, the HBCCA and the -ALL groups as designated, are shown below.

An example of an acid labile, lipophilic molecular conjugate (ALLMC),where the HBCCA group is paclitaxel having two -ALL groups, is depictedbelow:

In the above representative example of the acid labile molecularconjugate of paclitaxel, each of the -ALL¹ and -ALL² is independentlyhydrogen or an -ALL group as defined herein. For HBCCA groups havingmore than one hydroxyl groups, the inaccessible hydroxyl group or groupswhere the acid labile lipophilic group cannot be formed, then the groupthat is designated as an -ALL group(s) is hydrogen.

In another embodiment, there is provided a method of producing acidlabile, lipophilic molecular conjugates for use in treatment of cancerpatients. In one aspect, the method comprises a trans-ketalization ofsolketal (2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane) with an aldehydeor a ketone to form a compound of formula 4. Compound 4 may be condensedwith an acid halide (where X is a halide) to form the compound offormula 3. In one variation of the compound of the formula 3, thep-nitrophenoxy group may be replaced by a leaving group such as a2-halo-phenoxy, 2,4-halo-phenoxy, 2,4,6-trihalo-phenoxy,2,6-dihalo-phenoxy, wherein halo is selected from the group consistingof fluoro, chloro, bromo or iodo.

Condensation of 3 with a HBCCA (R—OH) provides the acid labile,lipophilic molecular conjugate of the cancer chemotherapeutic compound1, wherein R, R¹ and R² are as defined herein.

In another embodiment there is provided a method of preparing a compoundof the formula 2a comprising a condensation reaction of a HBCCA with anenol ether or vinyl ether to form a compound of the formula 2a:

wherein R—OH is the HBCCA, R³ is a C₂-C₂₃ alkyl, and X is hydrogen or ahalogen selected from Cl, Br or I.

In another embodiment, there is provided a method for concentrating acancer chemotherapeutic agent in selected target cells of a patientusing the acid labile, lipophilic molecular conjugates of the presentapplication in a nanoparticulate lipid emulsion resembling a LDLparticles or “pseudo-LDL particles”. In another embodiment, the methodcomprises administering to a patient a selected dose of atherapeutically effective amount of the acid labile, lipophilicmolecular conjugate of a cancer chemotherapeutic agent dissolved in thelipid core of the pseudo-LDL particles.

Also included in the above embodiments, aspects and variations are saltsof amino acids such as arginate and the like, gluconate, andgalacturonate. Some of the compounds of the invention may form innersalts or Zwitterions. Certain of the compounds of the present inventioncan exist in unsolvated forms as well as solvated forms, includinghydrated forms, and are intended to be within the scope of the presentinvention. Certain of the above compounds may also exist in one or moresolid or crystalline phases or polymorphs, the variable biologicalactivities of such polymorphs or mixtures of such polymorphs are alsoincluded in the scope of this invention. Also provided arepharmaceutical compositions comprising pharmaceutically acceptableexcipients and a therapeutically effective amount of at least onecompound of this invention.

Pharmaceutical compositions of the compounds of this invention, orderivatives thereof, may be formulated as solutions or lyophilizedpowders for parenteral administration. Powders may be reconstituted byaddition of a suitable diluent or other pharmaceutically acceptablecarrier prior to use. The liquid formulation is generally a buffered,isotonic, aqueous solution. Examples of suitable diluents are normalisotonic saline solution, 5% dextrose in water or buffered sodium orammonium acetate solution. Such formulations are especially suitable forparenteral administration but may also be used for oral administration.Excipients, such as polyvinylpyrrolidinone, gelatin, hydroxycellulose,acacia, polyethylene glycol, mannitol, sodium chloride, or sodiumcitrate, may also be added. Alternatively, these compounds may beencapsulated, tableted, or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols or water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.The amount of solid carrier varies but, preferably, will be betweenabout 20 mg to about 1 g per dosage unit. The pharmaceuticalpreparations are made following the conventional techniques of pharmacyinvolving milling, mixing, granulation, and compressing, when necessary,for tablet forms; or milling, mixing, and filling for hard gelatincapsule forms. When a liquid carrier is used, the preparation will be inthe form of a syrup, elixir, emulsion, or an aqueous or non-aqueoussuspension. Such a liquid formulation may be administered directly p.o.or filled into a soft gelatin capsule. Suitable formulations for each ofthese methods of administration may be found in, for example, Remington:The Science and Practice of Pharmacy, A. Gennaro, ed., 20th edition,Lippincott, Williams & Wilkins, Philadelphia, Pa.

These and other objects of the present invention will become morereadily appreciated and understood from a consideration of the followingdetailed description of the exemplary embodiments of the presentinvention when taken together with the accompanying drawings andfigures. The entire disclosures of all documents cited throughout thisapplication are incorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a graph of the stability of ART 273 when added to mouseplasma.

FIG. 2 depicts a graph of the stability of ART 273 when added to ratplasma.

FIG. 3 depicts a graph of the stability of ART 273 when added to humanplasma.

FIG. 4 depicts a graph of the stability of ART 488 when added to mouseplasma.

FIG. 5 depicts a graph of the stability of ART 488 when added to ratplasma.

FIG. 6 depicts a graph of the stability of ART 488 when added to humanplasma.

FIG. 7 depicts a graph of the stability of ART 488 in Liposyn® whenadded to mouse plasma.

FIG. 8 depicts a graph of the stability of ART 488 in Liposyn® whenadded to human plasma.

FIG. 9 depicts a graph of the stability of ART 198 when added to mouseplasma.

FIG. 10 depicts a graph of the stability of ART 198 when added to ratplasma.

FIG. 11 depicts a graph of the stability of ART 198 when added to humanplasma.

FIG. 12 depicts a graph of the stability of ART 489 when added to mouseplasma.

FIG. 13 depicts a graph of the stability of ART 489 when added to ratplasma.

FIG. 14 depicts a graph of the stability of ART 489 when added to humanplasma.

FIG. 15 depicts a graph of the stability of ART 489 in Liposyn® whenadded to mouse plasma.

FIG. 16 depicts a graph of the stability of ART 489 in Liposyn® whenadded to human plasma.

FIG. 17 depicts a graph of the stability of ART 467 when added to humanplasma.

FIGS. 18, 19 and 20 depict representative acid labile lipophilicmolecular conjugates.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

The following procedures may be employed for the preparation of thecompounds of the present invention. The starting materials and reagentsused in preparing these compounds are either available from commercialsuppliers such as the Aldrich Chemical Company (Milwaukee, Wis.), Bachem(Torrance, Calif.), Sigma (St. Louis, Mo.), or are prepared by methodswell known to a person of ordinary skill in the art, followingprocedures described in such references as Fieser and Fieser's Reagentsfor Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y.,1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supps.,Elsevier Science Publishers, 1989; Organic Reactions, vols. 1-40, JohnWiley and Sons, New York, N.Y., 1991; March J.: Advanced OrganicChemistry, 4th ed., John Wiley and Sons, New York, N.Y.; and Larock:Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

In some cases, protective groups may be introduced and finally removed.Suitable protective groups for amino, hydroxy, and carboxy groups aredescribed in Greene et al., Protective Groups in Organic Synthesis,Second Edition, John Wiley and Sons, New York, 1991. Standard organicchemical reactions can be achieved by using a number of differentreagents, for examples, as described in Larock: Comprehensive OrganicTransformations, VCH Publishers, New York, 1989.

General Procedure for Synthesis of Acid Labile, Lipophilic MolecularConjugates of Cancer Chemotherapeutic Agents.

Formation of Activated Intermediate Compounds:

Compounds suitable for use for forming acid labile, lipophilic molecularconjugates of cancer chemotherapeutic agents may be prepared accordingto the general methods disclosed herein. In one aspect, solketal isreacted with an alkyl aldehyde or a dialkyl ketone in the presence ofacid catalysis and an organic solvent to form the aldehyde solketal(acetal) derivative or the ketone solketal (ketal) derivative,respectively. According to the present method, 5-membered and 6-memberedcyclic acetals may be prepared and may be isolated in substantially pureform by chromatography. In one aspect, the solvent is toluene and thereaction is performed at an elevated temperature, such as about 60 to80° C. The acetal or ketal solketal derivative is subsequently activatedby a reaction with an acid halide, such as 4-nitrophenyl chloroformatein the presence of base catalysis to form the corresponding activatedderivative, such as the 4-nitrophenyl carbonate intermediate compound ofthe formula 3. In one aspect, the 4-nitrophenyl carbonate intermediatemay be condensed with a HBCCA to form the acid labile, lipophilicmolecular conjugate.

In another aspect solketal is first reacted with an acid halide such as4-nitrophenyl chloroformate in the presence of base catalysis to formsolketal nitrocarbonate which is subsequently reacted with an alkylaldehyde or a dialkyl ketone in the presence of acid catalysis and anorganic solvent to form the aldehyde solketal (acetal) derivative or theketone solketal (ketal) derivative of formula 3, respectively. In oneaspect, the solvent is toluene and the reaction is performed at RT. Inone aspect, the 4-nitrophenyl carbonate intermediate may be condensedwith a HBCCA to form the corresponding acid labile, lipophilic molecularconjugate.

In another aspect alcohol such as stearyl alcohol is reacted with vinylacetate in the presence of a transition metal catalyst such as[Ir(cod)Cl]₂ and a base additive such as Na₂CO₃ to form thecorresponding vinyl ether. In one aspect, the solvent is toluene and thereaction is performed at 100° C. In one aspect, the vinyl etherderivative may be condensed with a HBCCA to form the corresponding acidlabile, lipophilic molecular conjugate.

General Procedure for Synthesis of Alternative Acid Labile, LipophilicMolecular Conjugates of Cancer Chemotherapeutic Agents:

In one embodiment, the HBCCA may be reacted with the 4-nitrophenylcarbonate compound in the presence of a base, such as a catalytic amountof N,N-dimethyl-4-aminopyridine (DMAP) and pyridine, in an organicsolvent, such as dichloromethane (DCM) at room temperature (RT), to formthe desired acid labile, lipophilic molecular conjugate.

As shown in the scheme below, initial synthesis of activated acidlabile, lipophilic molecular conjugate intermediates have been obtainedby treating solketal with the aldehyde derived from the correspondingnatural fatty acid followed by reaction with 4-nitrophenylchloroformate.

However, this method result the formation of 5- and 6 memberedconjugates along with their corresponding syn/anti isomers. Althoughboth 5- and 6-membered acetals could act as lipophilic conjugateprecursors, 3 sets of regio- and stereo isomers were isolated in theacetal formation step. In one embodiment, the desired acetal may beisolated in substantially pure form by chromatography. An alternatereaction sequence for the preparation of the 5-membered acetal is shownbelow. This route provides the 5-membered acetal and provides a methodto access lipophilic conjugates of various candidate chemotherapeuticagents. The activated carbonate intermediate is further treated with thehydroxyl-bearing cancer chemotherapeutic agents to generate thecorresponding acid labile, lipophilic molecular conjugate prodrugs ofinterest.

Alternative acid labile, lipophilic molecular conjugates of cancerchemotherapeutic agents may be formed by reacting a HBCCA with an alkylvinyl ether in the presence of a halogenating agent, such as an NXS,such as N-bromosuccinimide (NBS) in DCM. In one aspect, the reactantsare combined in solution at low temperatures, such as about −78° C., andthe reaction is stirred and allowed to warm slowly to RT.

Other alternative acid labile, lipophilic molecular conjugates of cancerchemotherapeutic agents may be formed by reacting HBCCA with higheralkyl vinyl ethers (derived from natural fatty acids) in the presence ofan acid catalyst such as pyridinium para-toluene sulfonate (PPTS). Inone aspect, the reactants are combined in solution at RT to synthesizethe corresponding acid labile lipophilic acetal prodrug.

Formation of Acid-Labile Lipophilic Conjugates:

Method A: A solution of the 4-nitrophenyl carbonate-solketal conjugateof formula 3 (0.21 mmol) in anhydrous (anh.) dichloromethane (1 ml) wasadded to a solution of HBCCA (0.2 mmol) and DMAP (0.3 mmol) in anh.dichloromethane (2 ml) and the reaction mixture was stirred at RT undernitrogen atmosphere (N₂). The reaction progress was monitored byTLC/HPLC, upon completion, the reaction mixture was diluted withmethylene chloride (DCM), washed with NH₄Cl(s), water and brine. Theorganic layer was separated, dried over sodium sulfate and evaporated.The crude residue was purified by silica gel flash chromatography (SGFC)to obtain the conjugated prodrug.

Method B: To a solution of alkyl vinyl ether (1.2 mmol, 6 eq.) and HBCCA(0.2 mmol, 1 eq.) in anh. DCM (8 mL, 0.025M), NBS (1 mmol, 5 eq.) wasadded at −15° C. under N₂. The reaction mixture was stirred at −15° C.to 0° C. and the progress of the reaction was monitored by TLC/HPLC.Upon completion, the reaction mixture was diluted with DCM and thereaction mixture was washed with NaHCO₃(sat.), water and brine solution.Organic layer was dried over sodium sulfate and evaporated. The cruderesidue was purified by SGFC to yield the conjugated prodrug.

Method C: To a solution of alkyl vinyl ether (1.2 mmol, 6 eq.) and HBCCA(0.2 mmol, 1 eq.) in anh. DCM (8 mL, 0.025M), PPTS (0.02 mmol, 10 mol %)was added and the reaction mixture was stirred at RT under N₂. Thereaction progress was monitored by TLC/HPLC. Upon completion, thereaction mixture was diluted with DCM and the reaction mixture waswashed with NaHCO₃(sat.), water and brine solution. Organic layer wasdried over sodium sulfate and evaporated. The crude residue was purifiedby SGFC to yield the conjugated prodrug.

Characterization of Acid Labile Lipophilic Conjugates:

Acid labile lipophilic conjugates were characterized by a combination ofHPLC and High Resolution Mass Spectrometry. Specifics are provided witheach compound.

Preparation of ART 449

A solution of the 4-nitrophenyl carbonate of docosahexaenoic alcohol(0.5 g) in anh. DCM was added to a solution of ART 273 (0.522 g) andDMAP (0.140 g) in anh. DCM (18 mL) at RT under N₂ and stirred. Uponcompletion, the reaction was diluted with DCM, washed with saturatedammonium chloride solution (NH₄Cl(s)), water and brine. The organiclayer was separated, dried over sodium sulfate and evaporated. The cruderesidue was purified over silica gel to yield ART 449 as a white solid.−TOF MS: m/z 1003.4859 (M+CF₃C0₂)⁻

Preparation of ART 448

A solution of the 4-nitrophenyl carbonate of 5-hexen-1-ol (0.1 g) inanh. DCM was added to a solution of ART 273 (0.207 g) and DMAP (0.051 g)in anh. DCM (5 mL) at RT under N₂. Upon completion, the reaction wasdiluted with DCM, washed with NH₄Cl(s), water and brine. The organiclayer was separated, dried over sodium sulfate and evaporated. The cruderesidue was purified over silica gel to yield ART 448 as a white solid.−TOF MS: m/z 789.2928 (M+CF₃C0₂)⁻

Preparation of ART 473

Cyclohexyl vinyl ether (0.24 mL) was added to a solution of ART 273(0.230 g) and NBS (0.282 g) in anh. DCM (5 mL) at −78° C. under N₂. Uponcompletion, the solution was evaporated and the crude residue purifiedover silica gel to yield ART 473 as a white solid.

Preparation of ART 471

Tert-Butyl vinyl ether (0.24 mL) was added to a solution of ART 273(0.250 g) and NBS (0.307 g) in anh. DCM (5 mL) at −78° C. under N₂. Uponcompletion, the solution was evaporated and the crude residue purifiedover silica gel to yield ART 471 as a white solid.

Preparation of ART 472

Octadecyl vinyl ether (0.448 g) was added to a solution of ART 273(0.208 g) and NBS (0.255 g) in anh. DCM (5 mL) at −78° C. under N₂. Uponcompletion, the solution was evaporated and the crude residue purifiedover silica gel to yield ART 472 as a white solid.

Preparation of ART 470

Ethyl vinyl ether (0.11 mL) was added to a solution of ART 273 (0.150 g)and N-bromosuccinimide (NBS, 0.170 g) in anh. DCM (5 mL) at −78° C.under N₂. Upon completion, the solution was evaporated and the cruderesidue purified over silica gel to yield ART 470 as a white solid.

Preparation of ART 489

A solution of octadecyl solketal-4-nitrophenyl carbonate (0.750 g) inanh. DCM was added to a solution of ART 198 (0.754 g) and DMAP (0.238 g)in anh. DCM (30 mL) at RT under N₂. Upon completion, the reaction wasdiluted with DCM, washed with NH₄Cl(s), water and brine. The organiclayer was separated, dried over sodium sulfate and evaporated. The cruderesidue was purified over silica gel to yield ART 489 as a solid. −TOFMS: m/z 1031.4645 (M+CF₃C0₂)⁻

Preparation of ART 488

A solution of octadecyl solketal-4-nitrophenyl carbonate (0.53 g) inanh. DCM was added to a solution of ART 273 (0.507 g) and DMAP (0.168 g)in anh. DCM (30 mL) at RT under N₂. Upon completion, the reaction wasdiluted with DCM, washed with NH₄Cl(s), water and brine. The organiclayer was separated, dried over sodium sulfate and evaporated. The cruderesidue was purified over silica gel to yield ART 488 as a solid. −TOFMS: m/z 1003 4994 (M+CF₃C0₂)⁻

Preparation of ART 332

A solution of solketal-4-nitrophenyl carbonate (1.1 g) in anh. DCM wasadded to a solution of ART 273 (1.30 g) and DMAP (0.36 g) in anh. DCM(30 mL) at RT under N₂. Upon completion, the reaction was diluted withDCM, washed with NH₄Cl(s), water and brine. The organic layer wasseparated, dried over sodium sulfate and evaporated. The crude residuewas purified over silica gel to yield ART 332 as a white solid. −TOF MS:m/z 947.4601 (M+CF₃C0₂)⁻

Preparation of ART 441

DHA (0.2 g), DCC (0.157 g) and DMAP (0.006 g) were sequentially added toa solution of ART 273 (0.279 g) in anh. DCM (10 mL) at RT under N₂. Uponcompletion, the reaction was diluted with DCM, washed with NH₄Cl(s),water and brine. The organic layer was separated, dried over sodiumsulfate and evaporated. The crude residue was purified over silica gelto yield ART 441 (0.2 g) as a white solid.

Preparation of ART 467

A solution of octadecyl solketal-4-nitrophenyl carbonate (1.75 g) inanh. DCM was added to a solution of paclitaxel (2.59 g) and DMAP (0.557g) in anh. DCM (30 mL) at RT under N₂. Upon completion, the reaction wasdiluted with DCM, washed with NH₄Cl(s), water and brine. The organiclayer was separated, dried over sodium sulfate and evaporated. The cruderesidue was purified over silica gel to yield ART 467 as a white solid.−TOF MS: m/z 1306.5445 (M+CF₃C0₂)⁻

Preparation of ART 151

ART 151 was prepared by following the procedure as outlined in Method A.HPLC retention time 6.06, Method: Taxane conjugates_MKG4 (C18 column,MeOH/H₂O/THF 95/3/2 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5ml/min, 30° C., 14 min). +TOF MS: m/z 1239.6523 [M+18] (M+NH₄ ⁺)

Preparation of ART 152

ART 152 was prepared by following the procedure as outlined in Method B.HPLC retention time 8.21, Method: Taxane conjugates MKG4 (C18 column,MeOH/H₂O/THF 95/3/2 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5ml/min, 30° C., 14 min). +TOF MS: m/z 1228.5654 [M+1] (M+H⁺)

Preparation of ART 153

ART 153 was prepared by following the procedure as outlined in Method C.HPLC retention time 7.05, Method: Taxane conjugates_MKG4 (C18 column,MeOH/H₂O/THF 95/3/2 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5ml/min, 30° C., 14 min). +TOF MS: m/z 1150.6485 [M+1] (M+H⁺)

ART 161 was prepared by following the procedure as outlined in Method A.HPLC retention time 4.88, Method: Taxane conjugates_MKG6 (C18 column,MeOH/H₂O 95/5 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5 ml/min,30° C., 16 min). +TOF MS: m/z 1235.6276 [M+18] (M+NH₄ ⁺)

ART 207 was prepared by following the procedure as outlined in Method A.HPLC retention time 6.06, Method; Taxane conjugates_MKG17 (Synergycolumn, ACN/H₂O 60/40 to 100% ACN 10 min, 2 min 100% ACN, 230 nm, 1.5ml/min, 30° C., 15 min). +TOF MS: m/z 1220.6156 [M+1] and m/z 1237.6382[M+18] (M+NH₄ ⁺)

ART 156 was prepared by following the procedure as outlined in Method A.HPLC retention time 6.2, Method: Taxane conjugates_MKG4 (C18 column,MeOH/H₂O/THF 95/3/2 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5ml/min, 30° C., 14 min). +TOF MS: m/z 1176.6466 [M+1] and m/z 1193.6730[M+18] (M+NH₄ ⁺)

ART 162 was prepared by following the procedure as outlined in Method A.HPLC retention time 8.96, Method: Taxane conjugates_MKG16 (Synergycolumn, MeOH/H₂O 75/25 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5ml/min, 30° C., 15 min). +TOF MS: m/z 1189.6491 [M+18] and m/z 1172.6224[M+1] (M+H⁺)

ART 208 was prepared by following the procedure as outlined in Method A.HPLC retention time 7.4, Method: Taxane conjugates_MKG19 (Synergycolumn, ACN/H₂O 50/50 3 min, 80-100% ACN/H₂O 10 min, 2 min 100% ACN, 230nm, 1.5 ml/min, 30° C., 15 min). +TOF MS: m/z 1174.6306 [M+1] (M+H⁺)

ART 185 was prepared by following the procedure as outlined in Method C.HPLC retention time 6.42, Method: Taxane conjugates MKG15 (Synergycolumn, 70-100% ACN/H₂O 10 min, 100% ACN 2 min, 230 nm, 1.5 ml/min, 30°C., 15 min). +TOF MS: m/z 1104.6648 [M+1](M+H⁺) and m/z 1126.6447 [M+18](M+NH₄ ⁺)

ART 137 was prepared by following the procedure as outlined in Method C.HPLC retention time 10.63, Method: Taxane (C18 column, ACN/H₂O 50/50 to100% ACN 10 min, 2 min 100% ACNH, 230 nm, 1.5 ml/min, 30° C., 16 min)

ART 164 was prepared by following the procedure as outlined in Method A.HPLC retention time 7.73, Method: Taxane conjugates_MKG6 (C18 column,MeOH/H₂O 95/5 to 1000/% MeOH 10 min, 2 min 1000/MeOH, 230 nm, 1.5ml/min, 30° C., 16 min). +TOF MS: m/z 1255.7506 [M+18] (M+NH₄ ⁺)

ART 163 was prepared by following the procedure as outlined in Method A.HPLC retention time 7.56, Method: Taxane conjugates_MKG6 (C18 column,MeOH/H₂O 95/5 to 100% MeOH 10 min, 2 min 100% MeOH, 230 nm, 1.5 ml/min,30° C., 16 min). +TOF MS: m/z 1251.7233 [M+18] (M+NH₄ ⁺)

ART 209 was prepared by following the procedure as outlined in Method A.HPLC retention time 9.6, Method: Taxane conjugates_MKG18 (Synergycolumn, ACN/H₂O 80/20 10 min, 100% ACN 2 min, 230 nm, 1.5 ml/min, 30°C., 15 min). +TOF MS: m/z 1253.7505 [M+18](M+NH₄ ⁺)

Cytotoxicity of Specific Compounds:MTS Proliferation Assay Using SK-N-AS Cells

Day 1:

SK-N-AS cells were plated in appropriate growth medium at 5×10³ per wellin 100 μL in 96 well tissue culture plates, Falcon, one plate for eachdrug to be tested. Column 1 was blank; it contained medium, but nocells. The plates were incubated overnight at 37° C. in 5% CO₂ to allowattachment.

Day 2:

Drug diluted in culture media was added to the cells at a concentrationof 0.005 nM to 10 μM, in quadruplicate. After 48-72 hours of drugexposure, the MTS agent was added to all wells and incubated 1-6 hrs(37° C., 5% CO₂), depending on cell type, as per CellTiter 96® AQueousNon-Radioactive Cell Proliferation Assay (MTS), Promega. Plates wereprocessed using a Bio-Tek Synergy HT Multi-detection microtiter platereader at 490 nanometer wavelength and data were processed with KC4V.3software. Data plots of drug concentration vs. absorbance were plottedand the concentration resulting in 50% inhibition (IC₅₀) wasextrapolated for each of the tested compounds.

As summarized in Table 1, the IC₅₀ value for each tested compound in theSK-N-AS cell line was determined. The clinical comparator drug,paclitaxel, was included in the experiment to allow comparison of theresults of the candidate compounds to a clinically relevant standard inthe taxane class.

TABLE 1 IC₅₀ (nM) Values in SK-N-AS IC₅₀ (nM) Values in SK-N-AS (MDR−Neuroblastoma) Compound IC₅₀ ART 449 4.0 ± 0.5 ART 448 5.0 ± 0.7 ART 47312.6 ± 0.9  ART 471 261.6 ± 12   ART 470 349.1 ± 15   ART 488 0.33 ±0.1  ART 441 1.76 ± 0.5  ART 472 1.19 ± 0.5  ART 332 1.1 ± 0.5 ART 2732.0 ± 0.5 ART 467 273.9 ± 12   Paclitaxel 0.05 ± 0.01 MTT ProliferationAssay Using Paired MDR+ and MDR− Cell Lines

A second evaluation of the cytotoxicity of the acid labile, lipophilicmolecular conjugates was undertaken. The purpose of these experimentswas to compare the toxicity of the conjugates in multidrug resistantcells and their parental susceptible lines to test the hypothesis that asubset of these compounds would exhibit a similar level of toxicity inthe drug resistant lines as that observed in the parent susceptible cellline.

MTT-based cytotoxicity assays were performed using human cancer celllines and paired sublines exhibiting multidrug resistance. These linesincluded a uterine sarcoma line, MES-SA, and its doxorubicin-resistantsubline, MES-SA/Dx5. See W. G. Harker, F. R MacKintosh, and B. I. Sikic.Development and characterization of a human sarcoma cell line, MES-SA,sensitive to multiple drugs. Cancer Research 43: 4943-4950 (1983); W. G.Harker and B. I. Sikic. Multidrug (pleiotropic) resistance indoxorubicin-selected variants of the human sarcoma cell line MES-SA.Cancer Research 45: 4091 4096 (1985).

MES-SA/Dx5 exhibits a marked cross resistance to a number ofchemotherapeutic agents including vinblastine, paclitaxel, colchicine,vincristine, etoposide, dactinomycin, mitoxantrone and daunorubicin andmoderate cross resistance to mitomycin C and melphalan. However,resistance to bleomycin, cisplatin, carmustine, 5-fluorouracil ormethotrexate is not observed. MES-SA/Dx5 cells express high levels ofABCB1 (MDR1) mRNA and its gene product, the P-glycoprotein. MES-SA andMES-SA/Dx5 were purchased from the American Type Culture Collection(ATCC, Manassas, Va.).

The second set of cells tested, CCRF-CEM or simply CEM, were derivedfrom the blood of a patient with acute lymphoblastic leukemia. G. E.Foley, H. Lazarus, S. Farber, B. G. Uzman, B. A. Boone, and R E.McCarthy. Continuous culture of human lymphoblasts from peripheral bloodof a child with acute leukemia. Cancer 18: 522-529 (1965). The sublineCEM/VLB₁₀₀ was developed to be resistant to up to vinblastine at 100ng/ml. W. T. Beck, T. J. Mueller, and L. R. Tanzer. Altered surfacemembrane glycoproteins in Vinca alkaloid-resistant human leukemiclymphoblasts. Cancer Research 39: 2070-2076 (1979). Drug resistance isachieved by overexpression of the MDR1 gene. Resistance in the CEMsubline designated CEM/VM-1-5, however, is “atypical.” M. K. Danks, J.C. Yalowich, and W. T. Beck. Atypical multiple drug resistance in ahuman leukemic cell line selected for resistance to teniposide (VM-26).Cancer Research 47: 1297-1301 (1987). The classes of drugs included inthe “classic” multiple drug resistance phenotype are Vinca alkaloids,anthracyclines, epipodophyllotoxins and antibiotics. However, CEM/VM-1-5cells retain sensitivity to the Vinca alkaloids despite resistance andcross-resistance to etoposide, anthracyclines and mitoxantrone. Danks,M. K.; Schmidt, C. A.; Cirtain, M. C.; Suttle, D. P.; Beck, W. T.,Altered catalytic activity of and DNA cleavage by DNA topoisomerase IIfrom human leukemic cells selected for resistance to VM-26. Biochemistry1988, 27, 8861-8869. Resistance in CEM/VM-1-5 cells is effected by overexpression of the ABCC1 (MRP1) gene. CEM, CEM/VLB₁₀₀ and CEM/VM-1-5cells were obtained from Dr. WT Beck, University of Illinois at Chicago.

TABLE 2 Summary of Testing Concentrations in Paired Cell Lines Summaryof Testing Concentrations Compound Test Concentrations (ng/ml) ART 273200, 40, 8, 1.6, 0.32, 0.064 ART 198 5,000, 1,000, 200, 40, 8, 1.6 ART488 5,000, 1,000, 200, 40, 8, 1.6 ART 489 5,000, 1,000, 200, 40, 8, 1.6Paclitaxel 25,000 5,000, 1,000, 200, 40, 8, 1.6 Vinblastine Doxorubicin

TABLE 3 IC50 Results MESSA/ (nM) MESSA Dx5 Degree of CEM CEM/VLB₁₀₀Degree of CEM/VM-1-5 Degree of Compound (Hs) (MDR + Hs) resistance¹(HTL) (MDR + HTL) resistance² (MDR + HTL) resistance² ART 273 1.5 ± 0.77 ± 4 4.5 ± 0.7 2 ± 0 36 ± 20 18 ± 10 7 ± 2 3 ± 1 Q1 ART 488 1.5 ± 0.7  7 ± 5.7 4.3 ± 1.8 4 ± 0 34 ± 12 9 ± 4 8 ± 4 2 ± 1 Q1 prodrug ART 19847.5 ± 11   376 ± 110 7.9 ± 0.6 113 ± 53  7186 ± 1918 76 ± 52 308 ± 10 3 ± 1 Q2 ART 489 5.5 ± 3.5 17 ± 5.7 4.3 ± 3.8 21 ± 2  670 ± 71  33 ± 0 24 ± 4   1.2 ± 0.07 Q2 prodrug Paclitaxel 9 ± 7 19398 ± 204   3105 ±2416 <11/2 3029/1295 >275/648 <11/8 4 Vinblastine 1.1 ± 0.3 43 ± 12 38.5± 0.7    1 ± 0.8 227 ± 77  255 ± 127 1.3 ± 0.9  1.2 ± 0.07 Doxorubicin 297 49 14 2100 150 3060 219 Data are expressed as IC₅₀ values (nM).¹Calculated by dividing the IC₅₀ of the resistant lines by the IC₅₀ ofthe sensitive MES-SA cells. ²Calculated by dividing the IC₅₀ of theresistant lines by the IC₅₀ of the sensitive CEM cells. HTL means HumanT-Lymphoblastoid; Hs means Human sarcoma.

The observed cytotoxicity of the acid labile, lipophilic molecularconjugates demonstrates that they still possess the anti-cancer activitydesired for them to retain utility as potential chemotherapeutic agents.It is especially noteworthy that the apparent degree of resistanceexpressed by the resistant cell lines is diminished by 20 to 50% for theacid labile, lipophilic molecular conjugates. This was an unexpectedresult.

Stability of Acid Labile, Lipophilic Molecular Conjugates in Plasma:

The stability of the acid labile, lipophilic molecular conjugates tohydrolysis in plasma was evaluated to determine their potential torelease the active cancer chemotherapeutic agents into systemiccirculation and thereby cause general off target toxicity (“sideeffects”). The conjugates were incubated with plasma of mouse, rat andhuman origin.

HPLC grade Methanol from Fisher (Fair lawn, NJ, USA). Part No: A452-4(074833). HPLC grade Water from Fisher (Fair lawn, NJ, USA). Part No:W5-4 (073352). Drug-free mouse, rat and human plasmas were purchasedfrom Innovative Research Inc. (Southfield, Mich., USA). LIPOSYN® I.V.(30% soybean oil and egg yolk phospholipids, Abbott Laboratories) FatEmulsion from Hospira, Inc. (Lake Forest, Ill.).

Preparation of Plasma Incubations:

Each drug (ART 198, ART 273, ART 488 and ART 489) was prepared intriplicate in mouse, rat and human plasma individually at 10 pig/mlconcentration and vortexed for 1 minute and placed in a water bath at37° C. at a shake rate of 75 per minute. Samples were drawn at timepoints of 0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 210,240, 300, 360 and 480 minutes.

Analytical Method for ART 198, ART 273, ART 488 and ART 489 Analysis inPlasma:

Chromatographic separation of the compounds was performed on a WatersAcquity UPLC™ using a BEH C₁₈ column (1.7 μm, 2.1×50 mm). The mobilephase consisted of Methanol: 0.1% Formic acid (80:20). The flow rate was0.3 ml/min; the sample injection volume was 5 μL, resulting in a 3minute run time.

The MS instrumentation consisted of a Waters Micromass Quattro Micro™triple-quadrupole system (Manchester, UK). The MS system was controlledby a 4.0 version of MassLynx software. Ionization was performed in thepositive electrospray ionization mode. MS/MS conditions were thefollowing: capillary voltage 3.02 kV; cone voltage 50 v; extractorvoltage 5 v; and RF lens voltage 0.5 v. The source and desolvationtemperatures were 100° C. and 400° C. respectively, and the desolvationand cone gas flow were 400 and 30 L/hr, respectively.

The selected mass-to-charge (m/z) ratio transitions of the ART 198 usedin the selected ion monitoring (SIM) were: for ART 198, 617 (M+K)⁺, forART 273, 589 (M+K)⁺, for ART 488, 913 (M+Na)⁺, and for ART 489, 957(M+Na)⁺. The dwell time was set at 200 msec. MS conditions wereoptimized using direct infusion of standard solutions prepared inmethanol and delivered by a syringe pump at a flow rate of 20 μL/min.

Plasma Sample Preparation:

Samples of 100 μL were collected at time points of 0, 15, 30, 45, 60,75, 90, 105, 120, 135, 150, 165, 180, 210, 240, 300, 360 and 480 minutesrespectively and the reaction was terminated with methanol. In aseparate set of experiments the acid labile, lipophilic molecularconjugates were dissolved in a small amount of ethanol and diluted intoa lipid emulsion (Liposyn®) and added to mouse and human plasma beforeincubation and the hydrolysis of the conjugates was similarly measured.Collected plasma samples of 100 μL containing drug were placed inseparate Eppendorf micro centrifuge tubes for processing. Methanol (200μL) was added to extract the drug using the protein precipitationtechnique. The micro tubes were then vortex mixed for 10 minutes andcentrifuged for 15 minutes at a speed of 10,000 rpm (Eppendorf 5415Ccentrifuge). The supernatant was collected and filtered using a 0.45 μmfilter (Waters 13 mm GHP 0.45 μm) before analysis.

UPLC/MS/MS analysis of blank mouse, rat and human plasma samples showedno endogenous peak interference with the quantification of ART 198, ART273, ART 488 or ART 489.

The weighted linear least-squares (1/x) regression was used as themathematical model. The coefficient (r) for the compounds ranged from0.9925 to 0.9999. The calibration range was selected according to theconcentrations anticipated in the samples to be determined. The finalcalibration range was 10-12,500 ng/mL with a lower limit ofquantification of 10 ng/mL.

The repeatability and reproducibility bias (%) is within the acceptancelimits of ±20% at low concentration and ±15% at other concentrationlevels with RSD's of less than 5% at all concentrations evaluated.

The mean recoveries of the method were in the range of 86.22-99.83% atthree different concentrations of the test drugs from plasma. Theseresults suggested that there was no relevant difference in extractionrecovery at different concentration levels.

Incubations of ART 467 and Paclitaxel:

A 0.2 ml aliquot from 210.6 μg/ml stock solution of ART 467 was spikedinto 3.8 ml of human plasma preincubated for 15 min (37° C.) andincubated in a reciprocating water bath at 37° C. Samples were drawn at0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 hours.

Analytical Method for ART 467 and Paclitaxel (LiquidChromatography-Tandem Mass Spectrometry):

Chromatographic separation was carried out using an ACQUITY UPLC liquidchromatograph (Waters Corporation, Milford, Mass., USA) consisting of abinary pump, autosampler, degasser and column oven. A mobile phase ofmethanol-acetonitrile (50:50, v/v) was pumped at a flow-rate of 0.4ml/min through an ACQUITY UPLC BEH C₁₈ column (1.7 μm, 2.1×50 mm i.d.,Waters Corporation) maintained at 25° C. 10 μl of sample was injectedand the run time was 3.0 min. The LC elute was connected directly to anESCi triple-quadrupole mass spectrometer equipped with an electrosprayionization (ESI) ion source. The quadruples were operated in thepositive ion mode. The multiple reaction monitoring (MRM) mode was usedfor quantification using MassLynx version 4.1 software. Mass transitionsof m/z 876.2, 307.9; 882.2, 313.9; and 1216.5, 647.8 were optimized forpaclitaxel Na⁺ adduct, ¹³C6-paclitaxel adduct and ART 467 adductrespectively, with dwell time of 0.5 s. Nitrogen was used as nebulizinggas (30 l/h) and desolvation gas (300 l/h) with a desolvationtemperature at 250° C., and argon was collision gas. The capillaryvoltage was set at 3.5 kV, and cone voltage at 90 V. The sourcetemperature was set at 100° C.

Plasma Sample Preparation:

At the different time periods (0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24h), 200 μl aliquot of samples were taken and immediately added to 1.3 mlof cold TBME and subsequently 20 μl of internal standard stock solution(80.7 μg/ml in methanol) was added. Each tube was vortex mixed forapproximately 2 min and then centrifuged at 13000 rpm for 10 min. 1.0 mlof resultant supernatant was transferred to another tube and dried undera stream of nitrogen gas at 35° C. Each dried residue was reconstitutedwith 200 μl of methanol and vortex mixed for 0.5 min. Aftercentrifugation at 13000 rpm for 10 min, the supernatants weretransferred to HPLC autosampler vials, and 10 μl aliquot of each samplewas injected into LC-MS-MS.

Samples were collected at various times and the percent remaining of theacid labile, lipophilic molecular conjugate of the cancerchemotherapeutic agent was determined along with the percent of thechemotherapeutic agent released from the hydrolysis of the conjugate.The results are presented in table format and graphically.

Stability of Unconjugated ART 273 in Plasma:

The intrinsic stability of unconjugated ART 273 in mouse, rat and humanplasma was determined. Without reference to any particular kinetic modelit is seen that approximately 30%, 54%, and 67% of the initial ART 273remains after 480 minutes in mouse, rat and human plasma, respectively.

TABLE 4 Stability of ART 273 in Plasma at 37° C. ART 273 in ART 273 inART 273 in Mouse Plasma Rat Plasma Human Plasma Time, min ART 273 ART273 ART 273 0 100 100 100 15 81.1 87.9 97.2 30 76.0 84.9 96.5 45 68.482.9 94.6 60 65.4 78.9 93.5 75 62.7 71.8 93.5 90 54.8 69.7 92.2 105 53.866.1 89.4 120 49.8 64.6 87.0 135 46.8 64.3 86.6 150 44.0 61.8 85.5 16542.2 57.0 83.8 180 39.5 56.7 83.4 210 37.6 55.4 80.4 240 36.4 55.1 80.0300 33.8 54.7 73.2 360 31.5 54.5 69.3 480 30.1 53.9 66.7Stability of the ART 273 Conjugate, ART 488, in Plasma

The intrinsic stability of the ART 273 Conjugate, ART 488, in mouse, ratand human plasma was determined. Without reference to any particularkinetic model it is seen that approximately 36%, 33%, and 44% of theinitial ART 488 remains after 480 minutes in mouse, rat and humanplasma, respectively. Also without reference to any particular kineticmodel it is seen that the formation of ART 273 approximately equivalentto 36%, 32%, and 37% of the initial ART 488 is present after 480 minutesin mouse, rat and human plasma, respectively.

TABLE 5 Stability of ART 488 in Plasma at 37° C. ART 488 in ART 488 inART 488 in Mouse Plasma Mouse Plasma Human Plasma Time, ART ART ART ARTART ART min 488 273 488 273 488 273 0 100 0 100 0 100 0 15 91.2 3.3 90.72.2 90.8 1.2 30 85.7 7.9 80.6 8.7 89.1 7.3 45 81.3 10.7 79.8 10.1 87.89.3 60 75.0 11.3 78.3 11.7 87.9 10.3 75 73.2 12.2 78.0 12.4 87.9 11.2 9065.2 13.2 77.5 13.2 87.1 12.5 105 58.8 14.4 73.7 14.0 86.2 13.4 120 56.416.3 69.5 16.3 85.3 15.1 135 56.2 18.2 69.1 19.5 84.0 19.7 150 55.0 19.268.7 20.0 82.7 19.9 165 53.7 22.5 64.0 22.1 81.1 23.3 180 53.7 26.1 63.874.7 78.6 26.5 210 52.4 27.9 63.6 25.5 78.1 28.1 240 50.3 28.7 60.4 26.776.5 29.3 300 48.2 29.3 53.7 28.0 59.3 30.8 360 45.6 30.1 48.7 29.0 59.832.2 480 35.7 35.6 33.3 32.2 43.6 36.6Stability of the ART 273 Conjugate, ART 488, in Plasma when Added in aLipid Emulsion:

The intrinsic stability of the ART 273 Conjugate, ART 488, in mouse andhuman plasma was determined. Without reference to any particular kineticmodel it is seen that approximately 89% and 88% of the initial ART 488remains after 480 minutes in mouse and human plasma, respectively.

TABLE 6 Stability of ART 488 in Plasma at 37° C. When Added in a LipidEmulsion ART 488 ART 488 in Liposyn ® in Liposyn ® Time, in Mouse Plasmain Human Plasma min ART 488 ART 273 ART 488 ART 273 0 100 ND 100 ND^(a)15 98.7 ND 98.3 ND 30 98.2 ND 97.3 ND 45 97.4 ND 96.1 ND 60 96.9 ND 95.8ND 75 97.0 ND 95.3 ND 90 98.3 ND 95.6 ND 105 96.0 ND 94.6 ND 120 95.2 ND94.5 ND 135 93.8 ND 92.5 ND 150 93.1 ND 92.2 ND 165 92.9 ND 91.9 ND 18091.8 ND 91.0 ND 210 91.7 ND 91.0 ND 240 91.4 ND 90.7 ND 300 91.3 ND 90.7ND 360 90.0 ND 90.2 ND 480 88.5 ND 88.1 ND ^(a)ND = None detectedStability of Unconjugated ART 198 in Plasma:

The intrinsic stability of unconjugated ART 198 in mouse, rat and humanplasma was determined. Without reference to any particular kinetic modelit is seen that approximately 26%, 30%, and 34% of the initial ART 198remains after 480 minutes in mouse, rat and human plasma, respectively.

TABLE 7 Stability of ART 198 in Plasma at 37° C. ART 198 in ART 198 inART 198 in Time, Mouse Plasma Rat Plasma Human Plasma min ART 198 ART198 ART 198 0 100 100 100 15 96.8 95.8 99.3 30 94.0 84.0 99.1 45 85.566.0 94.9 60 82.0 55.7 94.6 75 72.6 54.4 93.1 90 66.9 54.2 89.9 105 63.254.0 87.0 120 59.2 52.1 68.5 135 57.4 48.9 66.4 150 51.9 48.9 61.1 16546.2 45.4 59.6 180 43.0 44.0 48.6 210 39.3 42.7 47.6 240 35.4 42.2 46.0300 32.4 34.3 44.4 360 28.8 30.1 39.6 480 25.9 30.1 34.2Stability of the ART 198 Conjugate, ART 489, in Plasma:

The intrinsic stability of the ART 198 Conjugate, ART 489, in mouse, ratand human plasma was determined. Without reference to any particularkinetic model it is seen that approximately 34%, 34%, and 66% of theinitial ART 489 remains after 480 minutes in mouse, rat and humanplasma, respectively. Also without reference to any particular kineticmodel it is seen that ART 198 equivalent to approximately 35%, 32%, and20% of the initial ART 489 is present after 480 minutes in mouse, ratand human plasma, respectively.

TABLE 8 Stability of ART 489 in Plasma at 37° C. ART 489 in ART 489 inART 489 in Mouse Plasma Rat Plasma Human Plasma Time, ART ART ART ARTART ART min 489 198 489 198 489 198 0 100 0 100 0 100 0 15 95.7 1.7 93.23.1 99.3 0.1 30 88.6 6.1 75.8 14.1 98.8 0.6 45 84.8 10.0 74.3 16.3 98.40.9 60 79.2 14.6 75.0 18.1 97.4 1.1 75 78.1 16.7 74.4 20.5 94.6 1.2 9070.1 18.2 74.4 20.8 93.7 2.4 105 68.0 20.3 73.7 21.4 93.0 3.2 120 64.121.3 69.9 21.9 91.9 5.1 135 63.2 22.1 68.5 22.3 91.7 6.5 150 59.4 25.167.3 22.9 90.9 7.2 165 54.7 26.4 63.0 23.6 90.4 8.5 180 51.6 27.6 63.024.5 90.1 9.6 210 50.3 29.7 62.7 25.0 89.0 12.3 240 47.5 32.0 61.7 25.386.7 14.2 300 41.1 34.1 55.4 26.1 84.1 16.3 360 38.1 34.3 48.2 28.0 78.719.5 480 34.0 34.7 34.3 32.3 65.9 20.4Stability of the ART 198 Conjugate, ART 489, in Plasma when Added in aLipid Emulsion:

The intrinsic stability of the ART 198 Conjugate, ART 489, in mouse andhuman plasma was determined. Without reference to any particular kineticmodel it is seen that approximately 73% and 77% of the initial ART 489remains after 480 minutes in mouse and human plasma, respectively.

TABLE 9 Stability of ART 489 in Plasma at 37° C. When Added in a LipidEmulsion ART 489 ART 489 in Liposyn ® in Liposyn ® Time, in Mouse Plasmain Human Plasma min ART 489 ART 198 ART 489 ART 198 0 100 ND 100 ND 1598.0 ND 98.4 ND 30 97.9 ND 93.9 ND 45 97.4 ND 92.7 ND 60 91.4 ND 88.2 ND75 90.3 ND 87.9 ND 90 87.9 ND 87.5 ND 105 80.7 ND 86.4 ND 120 80.4 ND86.4 ND 135 79.9 ND 84.7 ND 150 79.2 ND 84.6 ND 165 78.7 ND 83.7 ND 18078.2 ND 82.9 ND 210 75.6 ND 82.0 ND 240 74.7 ND 81.4 ND 300 73.7 ND 80.2ND 360 73.0 ND 78.2 ND 480 72.9 ND 76.6 NDStability of the Paclitaxel Conjugate, ART 467, in Plasma:

The intrinsic stability of the paclitaxel conjugate, ART 467, in humanplasma was determined. Without reference to any particular kinetic modelit is seen that approximately 41% of the initial ART 467 remains after1440 minutes in human plasma. Also without reference to any particularkinetic model it is seen that paclitaxel equivalent to approximately 16%of the initial ART 467 is present after 1440 minutes in human plasma.

TABLE 10 Stability of ART 467 in Human Plasma at 37° C. ART 467 in HumanPlasma Time, min ART 467 Paclitaxel 0 100.0 0.0 30 86.3 0.7 60 78.0 1.7120 76.0 2.7 180 75.0 3.8 240 73.7 5.5 360 72.8 8.2 480 70.5 10.3 60068.2 12.4 720 64.6 14.1 1440 41.3 15.5

Dissolution of the acid labile, lipophilic molecular conjugates ART 488and ART 489 in a lipid emulsion before addition to plasma enhanced thestability of the conjugate to hydrolysis by the plasma mediumdramatically (summarized in Table 11). That the acid labile, lipophilicmolecular conjugates remained within the lipid emulsion and did not“leak” into the plasma phase of the incubation is evident from the lackof release of the free drug from the conjugates. No detectableconcentrations of free drug could be observed in the incubations whereinthe conjugates were first dissolved in the lipid emulsion beforeaddition to the incubation medium (see Table 6 and Table 9).

TABLE 11 Drug Stabilization by Incorporation in a Lipid Emulsion % ofInitial Drug Remaining After 480 Minutes Mouse Rat Human Plasma PlasmaPlasma ART 273 30.1 53.9 66.7 ART 488 35.7 33.3 43.6 ART 488 in 88.5 NP88.1 Liposyn ART 198 25.9 30.1 34.2 ART 489 34.0 34.3 65.9 ART 489 in72.9 NP^(a) 76.6 Liposyn ^(a)NP = Experiment not performedEstimation of Maximum Tolerated Dose (MTD) of Acid Labile, LipophilicMolecular Conjugates in the Mouse:

Stock solutions of ART 198 and 273 and their respective acid labile,lipophilic molecular conjugates (ART 489 and ART 488, respectively) wereprepared in ethanol and then diluted into a lipid emulsion (INTRALIPID®,20% Soybean Oil, 1.2% egg yolk phospholipids, 2.25% glycerin and water)and injected intravenously into mice at various doses in milligrams perkilogram. The animals were observed daily for signs of toxicity and/ordeath for a period of 30 days. The MTD was defined as survival of thedosed mice for the full 30 day observation period.

The MTD of ART 198 was determined to be 4.0+/−1.0 mg/kg; the MTD of ART273 was determined to be 1.0+/−0.5 mg/kg; the MTD of ART 489 wasdetermined to be 3.1+/−1.0 mg/kg; and the MTD of ART 488 was determinedto be 4.0+/−0.5 mg/kg.

The observed similarity of MTD for ART 198 and its acid labile,lipophilic molecular conjugate ART 489, or in the case of ART 273, theincrease from an MTD of roughly 1 mg/kg for ART 273 to roughly 4 mg/kgfor its acid labile, lipophilic molecular conjugate ART 488 issurprising in light of their observed in vitro cytotoxicities. In invitro cytotoxicity evaluations, the acid labile, lipophilic molecularconjugates of ART 273 are routinely observed to be nearly an order ofmagnitude (10×) more potent than ART 273. The MTD determination resultssuggest that the acid labile, lipophilic molecular conjugates of cancerchemotherapeutic agent may be more useful for treating patients due toreduced toxicity.

What is claimed is:
 1. A pharmaceutical composition comprising an acidlabile lipophilic molecular conjugate (ALLMC) of the formula 1:

wherein: R is a 2′ hydroxyl residue of a paclitaxel, a docetaxel or anabeo-taxane compound; R¹ is hydrogen; R² is a C₅-C₂₂ alkyl; Y isselected from O, NR′ or S wherein R′ is hydrogen or C₁-C₆ alkyl; Z is Oor S; Q is O; and T is O; or an enantiomer, diastereoisomer or mixturesthereof; and a pharmaceutically acceptable salt thereof; wherein thepharmaceutical composition is formulated as a liquid formulation orsolution for parenteral administration.
 2. The pharmaceuticalcomposition of claim 1, wherein the liquid formulation is a lipidemulsion comprising INTRALIPID® for intravenous injection.
 3. Thepharmaceutical composition of claim 1, wherein the liquid formulation isa buffered, isotonic, aqueous solution.
 4. The composition of claim 1,wherein the liquid formulation further comprises a suitable diluentselected from the group consisting of normal isotonic saline solution,5% dextrose in water and buffered sodium or ammonium acetate solution.5. The pharmaceutical composition of claim 1, wherein the acid labilelipophilic molecular conjugate is of the formula:


6. The pharmaceutical composition of claim 1, wherein the acid labilelipophilic molecular conjugate of claim 1, where the conjugate is of theformula:


7. The pharmaceutical composition of claim 1, wherein the acid labilelipophilic molecular conjugate of claim 1, where the conjugate is of theformula:


8. The pharmaceutical composition of claim 1, wherein the acid labilelipophilic molecular conjugate of claim 1, where the conjugate is of theformula:


9. The pharmaceutical composition of claim 1, wherein the acid labilelipophilic molecular conjugate of claim 1, where the conjugate is of theformula:


10. The pharmaceutical composition of claim 1, wherein the acid labilelipophilic molecular conjugate of claim 1, where the conjugate is of theformula:


11. The pharmaceutical composition of claim 1, wherein the acid labilelipophilic molecular conjugate of claim 1, where the conjugate is of theformula:


12. The pharmaceutical composition of claim 1, wherein the acid labilelipophilic molecular conjugate of claim 1, where the abeo-taxaneconjugate is of the formula:


13. The pharmaceutical composition of claim 1, wherein the acid labilelipophilic molecular conjugate of claim 1, where the abeo-taxaneconjugate is of the formula:


14. The pharmaceutical composition of claim 1, wherein the acid labilelipophilic molecular conjugate of claim 1, where the abeo-taxaneconjugate is of the formula:


15. A method for the treatment of cancer in a patient comprisingadministering to the patient a therapeutically effective amount of acomposition of claim 1, to a patient in need of such treatment.
 16. Themethod of claim 15, wherein the cancer is selected from the groupconsisting of leukemia, neuroblastoma, glioblastoma, cervical,colorectal, pancreatic, renal and melanoma.
 17. The method of claim 15,wherein the cancer is selected from the group consisting of lung,breast, prostate, ovarian and head and neck.
 18. A method for reducingor eliminating the side effects of chemotherapy associated with theadministration of a cancer chemotherapeutic agent to a patient whencompared to the administration of a non-conjugated cancerchemotherapeutic agent, the method comprising administering to thepatient a therapeutically effective amount of a pharmaceuticalcomposition comprising an acid labile lipophilic molecular conjugate ofthe formula 1:

wherein: R is a 2′ hydroxyl residue of a paclitaxel, a docetaxel or anabeo-taxane compound; R¹ is hydrogen; R² is a C₅-C₂₂ alkyl; Y isselected from O, NR′ or S wherein R′ is hydrogen or C₁-C₆ alkyl; Z is Oor S; Q is O; and T is O; or an enantiomer, diastereoisomer or mixturesthereof; or a pharmaceutically acceptable salt thereof; wherein thepharmaceutical composition is formulated as a liquid formulation orsolution for parenteral administration.
 19. The method of claim 18,wherein the method provides a higher concentration of the cancerchemotherapeutic agent in a cancer cell of the patient, when compared tothe administration of a non-conjugated cancer chemotherapeutic agent.